cumA, a gene encoding a multicopper oxidase, is involved in Mn2+ oxidation in Pseudomonas putida GB-1.

Pseudomonas putida GB-1-002 catalyzes the oxidation of Mn2+. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designated cumA) encoding a protein homologous to multicopper oxidases. Addition of Cu2+ increased the Mn2+-oxidizing activity of the P. putida wild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu2+. A second open reading frame (designated cumB) is located downstream from cumA. Both cumA and cumB probably are part of a single operon. The translation product of cumB was homologous (level of identity, 45%) to that of orf74 of Bradyrhizobium japonicum. A mutation in orf74 resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the cumA mutant, suggesting that the cumA mutation may have a polar effect on cumB. This was confirmed by site-specific gene replacement in cumB. The cumB mutation did not affect the Mn2+-oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn2+ and that CumB is required for optimal growth of P. putida GB-1-002.

The cytochrome c maturation operon is involved in manganese oxidation in Pseudomonas putida GB-1.

A Pseudomonas putida strain, strain GB-1, oxidizes Mn2+ to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn2+ oxidation and/or secretion of the Mn2+-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn2+ oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn2+-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn2+ oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed.

Partial purification and characterization of manganese-oxidizing factors of Pseudomonas fluorescens GB-1.

The Mn(2+)-oxidizing bacterium Pseudomonas fluorescens GB-1 deposits Mn oxide around the cell. During growth of a culture, the Mn(2+)-oxidizing activity of the cells first appeared in the early stationary growth phase. It depended on the O2 concentration in the culture during the late logarithmic growth phase. Maximal activity was observed at an oxygen concentration of 26% saturation. The activity could be recovered in cell extracts and was proportional to the protein concentration in the cell extracts. The specific activity was increased 125-fold by ammonium sulfate precipitation followed by reversed-phase and gel filtration column chromatographies. The activity of the partly purified Mn(2+)-oxidizing preparation had a pH optimum of circa 7 and a temperature optimum of 35 degrees C and was lost by heating. The Mn(2+)-oxidizing activity was sensitive to NaN3 and HgCl2. It was inhibited by KCN, EDTA, Tris, and o-phenanthroline. Although most data indicated the involvement of protein in Mn2+ oxidation, the activity was slightly stimulated by sodium dodecyl sulfate at a low concentration and by treatment with pronase and V8 protease. By polyacrylamide gel electrophoresis, two Mn(2+)-oxidizing factors with estimated molecular weights of 180,000 and 250,000 were detected.

Enzymatic iron oxidation by Leptothrix discophora: identification of an iron-oxidizing protein.

An iron-oxidizing factor was identified in the spent culture medium of the iron- and manganese-oxidizing bacterial strain Leptothrix discophora SS-1. It appeared to be a protein, with an apparent molecular weight of approximately 150,000. Its activity could be demonstrated after fractionation of the spent medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A spontaneous mutant of L. discophora SS-1 was isolated which excreted neither manganese- nor iron-oxidizing activity, whereas excretion of other proteins seemed to be unaffected. Although the excretion of both metal-oxidizing factors was probably linked, the difference in other properties suggests that manganese and iron oxidation represent two different pathways. With a dot-blot assay, it was established that different bacterial species have different metal-oxidizing capacities. Whereas L. discophora oxidized both iron and manganese, Sphaerotilus natans oxidized only iron and two Pseudomonas spp. oxidized only manganese.

Oxidation of Manganese and Iron by Leptothrix discophora: Use of N,N,N',N'-Tetramethyl-p-Phenylenediamine as an Indicator of Metal Oxidation.

A new method for the quantification and characterization of manganese-oxidizing activity by spent culture medium of Leptothrix discophora SS-1 was developed. It is based on the formation of the dye Wurster blue from N,N,N',N'-Tetramethyl-p-phenylenediamine by oxidized manganese generated in the spent medium. The kinetic parameters thus obtained agreed well with data obtained with other methods. It was also possible to demonstrate iron oxidation by spent culture medium. The kinetics of the process and inhibition by enzyme poisons suggest that iron oxidation is enzymatically catalyzed. Probably two different factors are involved in manganese and iron oxidation.

Oxidation of Manganese and Formation of Mn(3)O(4) (Hausmannite) by Spore Coats of a Marine Bacillus sp.

Isolated spore coats of a marine Bacillus species were incubated in 25 mM MnCl(2) at pH 7.5. Manganese precipitates, formed on the coat surfaces, were analyzed by transmission electron microscopy, electron diffraction, and energy-dispersive X-ray spectroscopy. Initially, an amorphous manganese oxide was observed on the coats which recrystallized to hausmannite after prolonged incubation in the MnCl(2) solution. The spore coats catalyze the oxidation of Mn(II) and have no structural influence on the final mineral phase precipitated.

Manganese oxidation by spores and spore coats of a marine bacillus species.

Bacillus sp. strain SG-1 is a marine bacterial species isolated from a near-shore manganese sediment sample. Its mature dormant spores promote the oxidation of Mn to MnO(2). By quantifying the amounts of immobilized and oxidized manganese, it was established that bound manganese was almost instantaneously oxidized. When the final oxidation of manganese by the spores was partly inhibited by NaN(3) or anaerobiosis, an equivalent decrease in manganese immobilization was observed. After formation of a certain amount of MnO(2) by the spores, the oxidation rate decreased. A maximal encrustment was observed after which no further oxidation occurred. The oxidizing activity could be recovered by reduction of the MnO(2) with hydroxylamine. Once the spores were encrusted, they could bind significant amounts of manganese, even when no oxidation occurred. Purified spore coat preparations oxidized manganese at the same rate as intact spores. During the oxidation of manganese in spore coat preparations, molecular oxygen was consumed and protons were liberated. The data indicate that a spore coat component promoted the oxidation of Mn in a biologically catalyzed process, after adsorption of the ion to incipiently formed MnO(2). Eventually, when large amounts of MnO(2) were allowed to accumulate, the active sites were masked and further oxidation was prevented.

Manganese reduction by a marine Bacillus species.

Mature dormant spores of marine Bacillus sp. strain SG1 catalyze the oxidation of Mn(II) to MnO2. We report that vegetative cells of the same strain reduced MnO2 under low-oxygen conditions. The rate of reduction was a function of cell concentration. The process had a pH optimum of 7.5 to 8.0 and was inhibited by HgCl2, by preheating of the cells at 80 degrees C for 5 min, by antimycin A, and by N-heptyl-hydroxy-quinoline-N-oxide. At a nonlimiting O2 concentration, little MnO2 reduction was observed. Under these conditions, the process could be induced by the addition of NaN3. Spectrophotometric analysis of the Bacillus cells indicated the presence of type b and c cytochromes. Both types can be oxidized in situ by addition of MnO2 to the cells.